Methods to detect remnant cancer cells

ABSTRACT

Disclosed methods for detecting remnant cancer cells in a tissue sample.

FIELD OF THE INVENTION

This invention is directed to compounds, compositions and methods fordetecting remnant cancer cells in a tissue sample. The compoundsdescribed herein comprise a cancer cell targeting moiety or componentcovalently linked to a detection moiety or component that has beenmodified to be initially incapable of providing a signal. The targetingmoiety allows the compound to attach to and then preferentially beabsorbed by cancer cells as compared to normal cells. The detectionmoiety is preferably a fluorescent compound that has been modified to benon-fluorescent and is applied to a tissue sample. The detection moietyis specifically designed to be transformable from its non-fluorescent toits fluorescent structure by intracellular enzymes. Upon transformation,the detection moiety is once again capable of producing a signal. Theunique combination of such components permit the topical application toa tissue sample suspected of containing cancer cells and then signalingthe presence of any such cells.

STATE OF THE ART

Solid mass cancers afflict millions of patients in the US with over onemillion newly diagnosed cancers each year. These newly diagnosed cancersinclude breast cancer projected to be over 300,000 newly diagnosed casesin 2017; ovarian cancer projected to be over 22,000 newly diagnosedcases in 2017; lung cancer projected to over 220,000 newly diagnosedcases in 2017; brain cancer projected to be over 80,000 newly diagnosedmalignant and benign cancer cases in 2017; colorectal cancer projectedto over 130,000 newly diagnosed cases in 2017; to name a few. Many ofthese newly diagnosed cancers require surgery coupled with radiationand/or chemotherapy. However, notwithstanding some success, surgicalresection of solid mass tumors invariably results in remnant tumor cellsremaining in all too many patients. These remnant cancer cells lead totumor regrowth and/or metastasis and often require further surgicalprocedures in too many patients. Regardless, the presence of remnanttumor cells after resection invariably leads to less than desirableoutcomes in most if not all of such patients.

Although the art has addressed this problem with tumor imaging agents,many of such agents are restricted in their use to pre-surgicalintravenous administration. For example, U.S. Pat. No. 8,043,603discloses a folate-fluorescein conjugate that requires administration upto 24 hours or more before surgery in order to allow proper imaging ofthe tumor. These conjugates are fluorescent and the tumor image isacquired by the preferential uptake by cancer cells as compared tonormal cells. The folic acid portion of these molecules is used as atumor-targeting agent as many cancers will preferentially absorb folicacid as compared to normal cells. However, the fluorescent character ofthese compounds means that upon topical application, the entire surgicalsurface is fluorescent. Moreover, the data suggests that thenon-acylated versions of these compounds bleach when exposed to intenseUV light.

Moreover, the systemic administration of such conjugates renders itdifficult to distinguish remnant cancer cells from normal cells. Withoutbeing limited to any theory, it is believed that the bulk of the tumormass absorbs most of the imaging agent leaving the remnant cells withlittle opportunity to absorb sufficient amounts of the imaging agent tobe detectible. As the composition is administered systemically andnormal cells absorb the conjugate (albeit at lower amounts than cancercells), issues relating to systemic toxicity are of concern.

As noted previously, the use of fluorescent conjugates limits theirtopical utility as application to a tissue sample will result influorescence in all areas where the conjugate is applied. Steps toresolve fluorescence solely due to cancer cells become necessary inorder to avoid false positives.

Accordingly, there is an ongoing and critical need to accurately andtimely detect remnant tumor cells in a tissue sample such as a surgicalfield so as to remove as many of these cells prior to closing thepatient.

SUMMARY OF THE INVENTION

This invention is directed to the discovery that covalent conjugation ofcancer targeting compounds to pro-detection compounds provides fornon-signal generating conjugates suitable for identifying remnant cancercells remaining at or proximate to the surgical field after resection ofsolid mass tumors that over-express folic acid receptors. The targetingcomponent of these conjugates permits binding to and absorption of theconjugate by cancer cells. Upon absorption, the pro-detection componentof these conjugates is then converted intracellularly into a signalgenerating detection component the signal of which permitsidentification of remnant cancer cells.

Accordingly, in one embodiment, this invention is directed to a methodfor detecting cancer cells in a cellular mass suspected of containingcancer cells which method comprises:

-   -   contacting said mass with a conjugate comprising a tumor        targeting component and a pro-detection component so that the        conjugate selectively binds to and is then absorbed by cancer        cells whereupon the pro-detection component is intracellularly        converted into a detection component with a signaling        fingerprint;    -   generating and then measuring the signal obtained from the        detection component; and    -   correlating the presence or absence of said signal fingerprint        so as to assess the presence or absence of cancer cells.

In another embodiment, this invention is directed to a method fordetecting cancer cells in a cellular mass suspected of containing cancercells which method comprises contacting said cellular mass with apro-fluorescent compound of formula I:

wherein said compound selectively attaches to and is absorbed by cancercells and is then intracellularly converted into a fluorescent compoundwith a fluorescent fingerprint; measuring the fluorescence obtained byexposing the cellular mass to UV light; and correlating said emittedfluorescence to said fluorescent fingerprint so as to assess thepresence or absence of cancer cells,

further wherein R and R¹ are independently C₂-C₁₈ alkyl or cycloalkylgroups either or both optionally containing 1 to 8 heteroatoms selectedfrom the group consisting of oxygen, S(O)_(x), >NR³, —OP(O)_(y)H,—OS(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino, di(C₁-C₆ dialkyl)amino,—C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and oxo wherein y and z areindependently 1 or 2, and x is 0, 1 or 2;

R³ is hydrogen or C₁-C₆ alkyl; and

L is a linker of from 1 to 20 carbon atoms and 1 to 6 heteroatomsselected from the group consisting of oxygen, S(O)_(x), >NR³,—OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino,di(C₁-C₆dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and oxo whereiny and z are independently 1 or 2, and x is 0, 1 or 2;

or pharmaceutically acceptable salts and/or solvates thereof.

In another embodiment, this invention is directed to a method fordetecting cancer cells in a cellular mass suspected of containing cancercells which method comprises contacting said cellular mass with apro-fluorescent compound of formula II:

-   -   wherein said compound selectively attaches to and is absorbed by        cancer cells and is then intracellularly converted into a        fluorescent compound with a fluorescent fingerprint; measuring        the fluorescence obtained by exposing the cellular mass to UV        light; and correlating said emitted fluorescence to said        fluorescent fingerprint so as to assess the presence or absence        of cancer cells,    -   further wherein R and R¹ are independently C₂-C₁₈ alkyl or        cycloalkyl groups either or both optionally containing 1 to 8        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —OS(O)_(z)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2;    -   R³ is hydrogen or C₁-C₆ alkyl; and    -   L is a linker of from 1 to 20 carbon atoms and 1 to 6        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —OS(O)_(z)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2;    -   or pharmaceutically acceptable salts and/or solvates thereof.

In another embodiment, this invention is directed to a method fordetecting cancer cells in a cellular mass suspected of containing cancercells which method comprises contacting said cellular mass with apro-fluorescent compound of formula III:

-   -   wherein said compound selectively attaches to and is absorbed by        cancer cells and is then intracellularly converted into a        fluorescent compound with a fluorescent fingerprint; measuring        the fluorescence obtained by exposing the cellular mass to UV        light; and correlating said emitted fluorescence to said        fluorescent fingerprint so as to assess the presence or absence        of cancer cells,    -   R and R¹ are independently C₂-C₁₈ alkyl or cycloalkyl groups        either or both optionally containing 1 to 8 heteroatoms selected        from the group consisting of oxygen, S(O)_(x), >NR³,        —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino,        di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and        oxo wherein y and z are independently 1 or 2, and x is 0, 1 or        2;    -   R³ is hydrogen or C₁-C₆ alkyl; and    -   L is a linker of from 1 to 20 carbon atoms and 1 to 6        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —(O)S(O)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2;    -   or pharmaceutically acceptable salts and/or solvates thereof.

In one embodiment, this invention is directed to non-fluorescentfluorescein diester conjugates of formula I:

-   -   where:    -   R and R¹ are independently C₂-C₁₈ alkyl or cycloalkyl groups        either or both optionally containing 1 to 8 heteroatoms selected        from the group consisting of oxygen, S(O)_(x), >NR³,        —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino,        di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and        oxo wherein y and z are independently 1 or 2, and x is 0, 1 or        2;    -   R³ is hydrogen or C₁-C₆ alkyl; and    -   L is a linker of from 1 to 20 carbon atoms and 1 to 6        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2; and    -   or pharmaceutically acceptable salts and/or solvates thereof.

In one embodiment, this invention is directed to non-fluorescentfluorescein diester conjugates of formula II:

-   -   where:    -   R and R¹ are independently C₂-C₁₈ alkyl or cycloalkyl groups        either or both optionally containing 1 to 8 heteroatoms selected        from the group consisting of oxygen, S(O)_(x), >NR³,        —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino,        di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and        oxo wherein y and z are independently 1 or 2, and x is 0, 1 or        2;    -   R³ is hydrogen or C₁-C₆ alkyl; and    -   L is a linker of from 1 to 20 carbon atoms and 1 to 6        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2; and    -   or pharmaceutically acceptable salts and/or solvates thereof.

In one embodiment, this invention is directed to non-fluorescentfluorescein diester conjugates of formula III:

-   -   where:    -   R and R¹ are independently C₂-C₁₈ alkyl or cycloalkyl groups        either or both optionally containing 1 to 8 heteroatoms selected        from the group consisting of oxygen, S(O)_(x), >NR³,        —OP(O)_(y)H, —OS(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino,        di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH, —OH, and        oxo wherein x, y and z are independently 1 or 2, and x is 0, 1        or 2;    -   R³ is hydrogen or C₁-C₆ alkyl; and    -   L is a linker of from 1 to 20 carbon atoms and 1 to 6        heteroatoms selected from the group consisting of oxygen,        S(O)_(x), >NR³, —OP(O)_(y)H, —OS(O)_(z)H, —C(O)—, amino, C₁-C₆        alkylamino, di(C₁-C₆ dialkyl)amino, —C(O)O—, —C(O)NR³—, —C(O)OH,        —OH, and oxo wherein y and z are independently 1 or 2, and x is        0, 1 or 2;    -   or a pharmaceutically acceptable salt and/or solvate thereof.

In one embodiment, this invention provides for a pharmaceuticalcomposition comprising a pharmaceutically acceptable inert diluent and acompound of formula I, II, or III. In one embodiment, the pharmaceuticalcomposition is suitable for topical application onto a tissue sample.Suitable compositions for topical application include aqueouscomposition comprising sterile water and optionally a water soluble ormiscible co-solvent such as DMSO, ethanol, octanol, and the like. Whenemployed as a co-solvent, the water preferably comprises from about 50to about 95 weight percent of the aqueous composition and the co-solventcomprises from about 5 to about 50 weight percent of the aqueouscomposition.

In one embodiment, R, R¹ and L are selected to provide sufficient watersolubility to the compound of formula I, II, or II so that thesecompounds can be formulated into an aqueous composition as describedherein for application to a tissue sample. In one embodiment, R and/orR¹ is an ester formed from a carboxyl containing sugar.

In one embodiment, this invention is directed to a method for detectingcancer cells in a cellular composition suspected of containing suchcells wherein said method comprises:

-   -   preparing a sterile aqueous composition of a water-soluble        fluorescein diester of formula I, II or III wherein said aqueous        composition optionally comprises up to 50 percent by weight of a        water soluble or miscible and biologically compatible        co-solvent;    -   applying said composition to a cellular composition suspected of        containing cancer cells;    -   maintaining said contact for a sufficient period of time to        permit absorption of said diester and intracellular deacylation        thereof by said cancer cells if present;    -   optionally lavaging said surface with a sterile aqueous solution        free of any of said diester(s); and    -   assessing the presence of intracellular fluorescence in said        cellular mass as an indicia of the presence of cancer cells in        said mass.

Specific compounds as well as salts and solvates of formula I of thisinvention include the following (left side of L is attached to folicacid):

I

# R/R¹ R² L 1 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— H

2 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— CH₃

3 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— H—NHCH₂CH₂CH₂(OCH₂CH₂)₃CH₂NHC(═S)NH— 4 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— H

5 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— H

6 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃CH₂C(O)O— H

7 CH₃O₂C(CH₂CH₂O)₃C(O)(CH₂CH₂O)₃C(O)O—CH₃O₂C(CH₂CH₂O)₃C(O)(CH₂CH₂O)₃C(O)O— H

8 CH₃O(CH₂CH₂O)₂CH₂C(O)O—/ CH₃O(CH₂CH₂O)₂CH₂C(O)O— H

Specific compounds as well as salts and solvates of formula I of thisinvention include the following (left side of L is attached to pteroicacid):

II

No. R/R¹ L  9 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—

10 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—

11 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—

12 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—

13 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O— CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—

14 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)O—/ CH₃CH₂C(O)O—

15 CH₃O₂C(CH₂CH₂O)₃C(O)(CH₂CH₂O)₃C(O)O—

16 CH₃O(CH₂CH₂O)₄CH₂CH₂C(O)O—/ CH₃O(CH₂CH₂O)₄CH₂CH₂C(O)O—

Specific compounds and salts/solvates of formula I of this inventioninclude the following (left side of L is attached to the nitrogen ofglucosamine):

III

Compound No. R/R¹ L 17 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)— —C(═S)NH— 18 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)— —CH₂CH₂CH₂NHC(═S)NH— 19CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)— —CH₂NHC(═S)NH— 20CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)— —CH₂CH₂NHC(═S)NH—21 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/ CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)——CH₂CH₂NHC(═O)NH— 22 CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—/ CH₃CH₂C(O)— —C(═S)NH—23 CH₃O₂C(CH₂CH₂O)₃C(O)(CH₂CH₂O)₃C(O)— —C(═S)NH— 24CH₃O(CH₂CH₂O)₄CH₂CH₂C(O)—/ CH₃O(CH₂CH₂O)₄CH₂CH₂C(O)— CH₂NHC(═S)NH— 25

—CH₂CH₂NHC(═S)NH— Glucuronic acid derivative

In one preferred embodiment, the compounds of this invention whenconverted to their fluorescent form are capable of maintaining afluorescent signal for at least 5 minutes and preferably at least 10minutes after exposure to UV light. In addition, the resultingfluorescent compounds of this invention preferably do not efflux fromthe cells back into the solution.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the intra-cellular generation of fluorescence ofcancer cells that have absorbed a conjugate of this invention and thenintracellularly deacylated said conjugate.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, this invention provides for compounds, compositions andmethod for detecting remnant cancer cells in a surgical field afterresection of a solid mass tumor. However, prior to providing a detaileddescription of the several aspects of this invention, the followingterms will first be defined.

Definitions

As used herein and in the appended claims, singular articles such as “a”and “an” and “the” and similar referents in the context of describingthe elements (especially in the context of the following claims) are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the claims unless otherwise stated. No language in the specificationshould be construed as indicating any non-claimed element as essential.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, C¹⁴, P³²and S³⁵ are thus within the scope of this invention. Procedures forinserting such labels into the compounds of this invention will bereadily apparent to those skilled in the art based on the disclosureherein.

The term “cancer”, “solid mass cancers or tumors” and “cancer cells”refer to cancer cells that form solid masses and as such blood bornecancers are not included within these terms.

It is also generally recognized that in many but not all cancers, theirrapid growth requires preferential uptake of folic acid (a nucleosideprecursor). As such, the term “folic acid requiring cancer cells” referto those cancer cells that over-express the folic acid receptor which issome cases is up to as many as 8-10 times that of normal cells. Cancercells recognized to require folic acid and, therefore, have an excess offolic acid receptors include, but are not limited to, epithelial cancercells, ovarian cancer cells, cervical cancer cells, breast cancer cells,lung cancer cells, kidney cancer cells, colorectal cancer cells, andbrain cancer cells. Likewise, it is generally recognized that in orderto keep up with the high nutritional and energy needs of a malignanttumor, cancer cells show a 20- to 30-fold higher rate of glucose uptakeand glycolysis compared with normal cells. Hence, the use of sugars thatare recognized by the glucose receptors on malignant tumor cells provideone route to conjugate the pro-detectible label and have the conjugateformed thereby preferentially taken up by these cancer cells.Accordingly, with compounds of formula III as well as those having adifferent sugar components that are recognized by one or more of theglucose receptors (e.g., glucosamine and fructose), all solid mass tumorcells that require high glucose uptake are suitable for identificationby the methods of this invention.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

“Substituted alkyl” refers to an alkyl group having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, acyloxy, aryl,substituted aryl, arylene, substituted arylene, heteroarylene,substituted heteroarylene, aryloxy, substituted aryloxy, carboxyl,carboxyl ester, cyano, cycloalkyl, cycloalkylene, substitutedcycloalkyl, substituted cycloalkylene, cycloalkyloxy, substitutedcycloalkyloxy, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heterocyclic, substitutedheterocyclic, heterocyclyloxy, substituted heterocyclyloxy, nitro, oxo,and SO₃H.

“Alkoxy” refers to the group —O-alkyl wherein alkyl is defined herein.Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

“Substituted alkoxy” refers to the group —O-(substituted alkyl).

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, aryl-C(O)—,substituted aryl-C(O)—, heteroaryl-C(O)—, substituted heteroaryl-C(O)—,heterocyclic-C(O)—, and substituted heterocyclic-C(O)—. Acyl includesthe “acetyl” group CH₃C(O)—.

“Acylamino” refers to —NR⁴⁷C(O)alkyl, —NR⁴⁷C(O)substituted alkyl,—NR⁴⁷C(O)cycloalkyl, —NR⁴⁷C(O)substituted cycloalkyl, —NR⁴⁷C(O)aryl,—NR⁴⁷C(O)substituted aryl, —NR⁴⁷C(O)heteroaryl, —NR⁴⁷C(O)substitutedheteroaryl, —NR⁴⁷C(O)heterocyclic, and —NR⁴⁷C(O)substituted heterocyclicwherein R⁴⁷ is hydrogen or alkyl.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—, substitutedcycloalkyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O.

“Aminocarbonyl” refers to the group —C(O)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group.

“Aminocarbonylamino” refers to the group —NR⁴⁷C(O)NR⁵⁰R⁵¹ where R⁴⁷ ishydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic, andwhere R⁵⁰ and R⁵¹ are optionally joined together with the nitrogen boundthereto to form a heterocyclic or substituted heterocyclic group.

“Aminocarbonyloxy” refers to the group —(O)—C(O)NR⁵⁰R⁵¹ where R⁵⁰ andR⁵¹ are independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclic, and substituted heterocyclic and where R⁵⁰ and R⁵¹ areoptionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, aminocarbonyl,aminocarbonylamino, aminocarbonyloxy, aryl, substituted aryl, aryloxy,substituted aryloxy, carboxyl, carboxyl ester, cyano, cycloalkyl,substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, halo,hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substitutedheteroaryloxy, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, nitro, and SO₃H.

“Arylene” refers to a divalent aromatic carbocyclic group of from 6 to14 carbon atoms having a single ring or multiple condensed rings.“Substituted arylene” refers to an arylene having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents as defined foraryl groups.

“Heteroarylene” refers to a divalent aromatic group of from 1 to 10carbon atoms and 1 to 4 heteroatoms selected from the group consistingof oxygen, nitrogen and sulfur within the ring. “Substitutedheteroarylene” refers to heteroarylene groups that are substituted withfrom 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituentsselected from the group consisting of the same group of substituentsdefined for substituted aryl.

“Aryloxy” refers to the group —O-aryl, where aryl is as defined herein,that includes, by way of example, phenoxy and naphthoxy.

“Substituted aryloxy” refers to the group —O-(substituted aryl) wheresubstituted aryl is as defined herein.

“Carbonyl” refers to the divalent group —C(O)—which is equivalent to—C(═O)—.

“Carboxyl” or “carboxy” refers to —COOH or salts thereof.

“Carboxyl ester” or “carboxy ester” refers to the group —C(O)(O)-alkyl,—C(O)(O)-substituted alkyl, —C(O)(O)-aryl, —C(O)(O)-substituted-aryl,—C(O)(O)—cycloalkyl, —C(O)(O)-substituted cycloalkyl,—C(O)(O)-heteroaryl, —C(O)(O)—substituted heteroaryl,—C(O)(O)-heterocyclic, and —C(O)(O)-substituted heterocyclic.

“Oxycarbonyl esters” refer to the group “acyloxy” as defined above.

In a preferred embodiment, such ester groups contain 2 to 18 carbonatoms and optionally contain from 1 to 6 heteroatoms selected from thegroup consisting of oxygen, S(O)_(x), NR³, —OP(O)_(y)H, —OS(O)_(z)Hwherein y and z are independently 1 or 2, and x is 0, 1 or 2 wherebysuch substitutions impart improved water solubility or maintainwater-solubility for the compound to which they are attached.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. The fused ring can be an aryl ring provided that thenon-aryl part is joined to the rest of the molecule. Examples ofsuitable cycloalkyl groups include, for instance, adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to acycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3substituents selected from the group consisting of oxo, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aryl, substitutedaryl, aryloxy, substituted aryloxy, carboxyl, carboxyl ester, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heterocyclic, substitutedheterocyclic, heterocyclyloxy, substituted heterocyclyloxy, nitro, andSO₃H.

“Cycloalkylene” refers to a divalent cycloalkyl group and “substitutedcycloalkylene” refers to a divalent substituted cycloalkyl group.

“Cycloalkyloxy” refers to —O-cycloalkyl.

“Substituted cycloalkyloxy refers to —O-(substituted cycloalkyl).

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Certain non-limiting examplesinclude pyridinyl, pyrrolyl, indolyl, thiophenyl, oxazolyl, thizolyl,and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy” refers to the group —O-(substitutedheteroaryl).

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen.Heterocycle encompasses single ring or multiple condensed rings,including fused bridged and spiro ring systems. In fused ring systems,one or more the rings can be cycloalkyl, aryl, or heteroaryl providedthat the point of attachment is through a non-aromatic ring. In oneembodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic groupare optionally oxidized to provide for the N-oxide, sulfinyl, orsulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5 or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

“Heterocyclyloxy” refers to the group —O-heterocycyl.

“Substituted heterocyclyloxy” refers to the group —O-(substitutedheterocycyl).

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, furan, thiophene, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

A substituted ring can be substituted with one or more fused and/orspiro cycles. Such fused cycles include a fused cycloalkyl, a fusedheterocyclyl, a fused aryl, a fused heteroaryl ring, each of which ringscan be unsubstituted or substituted. Such spiro cycles include a fusedcycloalkyl and a fused heterocyclyl, each of which rings can beunsubstituted or substituted.

“Optionally substituted” refers to a group selected from that group anda substituted form of that group. Substituents are such as those definedhereinabove. In one embodiment, substituents are selected from C₁-C₁₀ orC₁-C₆ alkyl, substituted C₁-C₁₀ or C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl, C₃-C₈ cycloalkyl, C₂-C₁₀ heterocyclyl, C₁-C₁₀heteroaryl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl,substituted C₆-C₁₀ aryl, substituted C₃-C₈ cycloalkyl, substitutedC₂-C₁₀ heterocyclyl, substituted C₁-C₁₀ heteroaryl, halo, nitro, cyano,—CO₂H or a C₁-C₆ alkyl ester thereof.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“alkoxycarbonylalkyl” refers to the group (alkoxy)-C(O)-(alkyl)-.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. In such cases, the maximumnumber of such substituents is three. That is to say that each of theabove definitions is constrained by a limitation that, for example,substituted aryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

It is understood that the substituents set forth herein and in the abovedefinitions are not intended to include impermissible substitutionpatterns (e.g., methyl substituted with 5 fluoro groups). Suchimpermissible substitution patterns are well known to the skilledartisan.

“Tautomer” refer to alternate forms of a compound that differ in theposition of a proton, such as enol/keto and imine/enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N— moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. In thecase of fluorescein, it exists in two tautomeric forms as shown belowand each are covered by the term “fluorescein”:

As used herein, the term stereochemically pure denotes a compound whichhas 80% or greater by weight of the indicated stereoisomer and 20% orless by weight of other stereoisomers. In a further embodiment, thecompound of Formula (I), (II), or (III) has 90% or greater by weight ofthe stated stereoisomer and 10% or less by weight of otherstereoisomers. In a yet further embodiment, the compound of Formula (I),(II), or (III) has 95% or greater by weight of the stated stereoisomerand 5% or less by weight of other stereoisomers. In a still furtherembodiment, the compound of formula (I), (II), or (III) has 97% orgreater by weight of the stated stereoisomer and 3% or less by weight ofother stereoisomers.

All terms not defined herein have their conventional definitions.

Synthesis

The compounds of this invention can be prepared from readily availablestarting materials using the following general methods and procedures.It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and P. G. M. Wuts, Protecting Groups inOrganic Synthesis, Third Edition, Wiley, New York, 1999, and referencescited therein.

If the compounds of this invention contain one or more chiral centers,such compounds can be prepared or isolated as pure stereoisomers, i.e.,as individual enantiomers or d(l) stereomers, or asstereoisomer-enriched mixtures. All such stereoisomers (and enrichedmixtures) are included within the scope of this invention, unlessotherwise indicated. Pure stereoisomers (or enriched mixtures) may beprepared using, for example, optically active starting materials orstereoselective reagents well-known in the art. Alternatively, racemicmixtures of such compounds can be separated using, for example, chiralcolumn chromatography, chiral resolving agents and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce orSigma (St. Louis, Mo., USA). Others may be prepared by procedures, orobvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley, and Sons, 5^(th) Edition, 2001)and Larock's Comp. Organic Transformations (VCH Publishers Inc. 1989).

In one embodiment, the compounds of this invention can be prepared from5- or 6- thioisocyanate fluorescein by first acylating both phenolichydroxyl groups as shown below:

where R and R¹ are as defined herein. Compound 2 defines a class ofnovel intermediates within the scope of this invention.

In particular, compound 1 (5-isothiocyanate fluorescein) is contactedwith at least two equivalents or more of a carboxyl acid chloride orother halide under esterifying conditions wherein each R and R¹ togetherwith the carbonyl group —C(O)— is an acyl group as defined herein. Thereaction is preferably conducted in a suitable aprotic inert diluentsuch as chloroform, ethyl acetate, methylene chloride and the like at atemperature of from about 0° to about 60° C. for a period of timesufficient to substantially complete the reaction. A suitable amount ofa base such as triethylamine, diisopropylethylamine, and the like istypically added to scavenge the acid generated during the reaction. Theproduct is typically isolated and purified by conventional techniquessuch as precipitation, crystallization, chromatography and the like.

Alternatively, as is well known in the art, compound 2 can be preparedby reaction with the corresponding acid anhydride [RC(O)OC(O)R] or mixedacid anhydride [RC(O)OC(O)R¹] by methods well known in the art. Inaddition, the isothiocyanate groups can be replaced with isocyanate,amino and haloacetamido groups all of which are commercially availableas a fluorescein derivative and have the general structure:

where R and R¹ are as defined herein and W is a substituent selectedfrom the group consisting of amino, haloacetamido, isocyanate, andthioisocyanate.

It is understood that the acyl groups selected are such that theresulting diester is soluble in an aqueous composition as definedherein. By “soluble”, it is meant that the compound has sufficientsolubility to provide detectible evidence of the presence of cancercells when the fluorescein moiety is deacylated. In one preferredembodiment, solubility of at least 0.1 mg/mL in the aqueous compositionat 25° C. and preferably at least 0.5 mg/mL is desireable.

Acyl groups that impart water solubility to the fluorescein diesters ofthis invention preferably comprise from 2 to 18 carbon atoms and from 1to 12 heteroatoms such as oxygen. Suitable hydroxyl acyl groups include,by way of example only, HOC(O)CH₂CH₂C(O)OCH₂CH₂OCH₃ (compound 3),CH₃OCH₂CH₂OCH₂CH₂OCH₂C(O)OH (compound 4), andCH₃OCH₂CH₂OCH₂CH₂OCH₂CH₂OCH₂CH₂C(O)OH (compound 5). Compound 3 is aknown compound prepared by reaction of succinic anhydride withapproximately 1 equivalents of 2-methoxyethanol. The reaction proceedsvia ring opening of the anhydride to provide for3-(2-methoxyethoxy)carbonylproprionic acid. Compounds 4 and 5 arecommercially available from Sigma-Aldrich, St. Louis, Mo., USA.

Synthetic schemes for the linkage of such soluble fluoresceinderivatives to folic acid first proceeds via coupling of a linker tofolic acid as shown in the following exemplary reaction scheme using adiamine linker:

In the case of folic acid, it is sometimes desirable to start with thefolic acid anhydride. That compound can be prepared as per theprocedures set forth in Guaranga, et al., Bioconjugate Chemistry, 2012,23:84-96 which are incorporated herein by reference in their entirety.

In one embodiment, folic acid anhydride can be reacted with, e.g.,NH₂-L-NHPg where Pg is a protecting group, L is a linker. One example ofa suitable linker is >CH—R¹⁰ where R¹⁰ an amino acid side chain. Anotherexample of a suitable linker is a polyoxyalkylene group of the formula-(alkylene—(O))_(p)— where p is from 1 to 20, and the like. Otherexamples of compounds suitable for linking the folic or pteroic acid tofluorescein diesters include NH₂-(alkylene—(O))_(p)—H, 4—H₂NCH₂-aniline,2-aminoethanol, and the like as illustrated in the reaction below.

The reaction proceeds under conventional amidation conditions and theresulting product, compound 6, is then isolated and purified byconventional methods or, alternatively used in the next step(s) withoutisolation and purification.

As to the next steps, the protecting group (Pg) is removed byconventional methods and the amino group is then free to react with theisothiocyanate group of compound 2. This reaction proceeds underconventional conditions and results in covalent linkage of the folicacid component or moiety to the fluorescein diester component or moietythrough a thiourea bond.

Alternatively, 5-isothiocyanate fluorescein diesters can be reacted withpteroic acid in a similar manner as described above. In this reaction,the carboxyl group of pteroic acid is amidated with the NH₂-L-NHPg toform an amido-L-NHPg substituent. Again, the protecting group (Pg) isremoved by conventional means. The resulting amino group then is reactedwith the isothiocyanate group of the fluorescein diester to provide forthe thiourea linkage of the pteroic acid component or moiety to thefluorescein diester component or moiety. Still further, glucose aminecan be reacted directly with the 5-chloroacetamido group of thefluorescein diester as shown below:

Due to the potential for deacylation by the amino group of the glucoseamine, the reaction is preferably conducted at a temperature of from−40° C. to about 20° C. The reaction is conducted in an inert solventsuch as methylene chloride, chloroform, ethyl acetate, toluene, DMF andthe like in the presence of a suitable base such asdiisopropylethylamine to scavenge the acid generated. Alternatively, thereaction can be conducted in pyridine. The reaction continues untilsubstantial completion which typically occurs within 1 to 24 hours. Theproduct is recovered and purified by conventional techniques such aschromatography, crystallization, HPLC and the like.

Alternatively, a hydroxyl-L-amine of the formula NH₂-L-OH can be used inthe reactions above with the isothiocyanate fluorescein diester. Theamino group of the hydroxyl-L-amine will react with the carboxyl groupon pteroic acid or with a carboxyl group on folic acid to provide forthe corresponding amide alcohol [—C(O)NH-L-OH]. The alcohol then reactswith the isothiocyanate group to provide for a thiocarbamate linkage ofthe folic acid or pteroic acid moiety or component to the fluoresceindiester moiety or component. In these reactions, L is a linker asdefined above.

Preferred compounds of the formula NH₂-L-OH and NH₂-L-NH₂ include thosewhere L is a polyoxyalkylene or an alkylene group. Compounds such asNH₂-L-OH or NH₂-L-NH₂ are commercially available as Jeffamines (HuntsmanCorp.) or from Sigma-Aldrich, supra.

Other reaction schemes to couple the reactive functionalities of thecompounds of this invention with cellular targeting agents are wellknown. For example, amino groups on the lysine residues of antibodiescan react with a —COOH, —N═C═S or —N═C═O group as per above. Carboxylresidues on aspartic and glutamic acid can react with the amino group on5-amino fluorescein diesters.

Methods

The compounds of this invention are non-fluorescent by virtue of lockingthe fluorescein into a non-fluorescent tautomeric structure due todiesterification of both phenolic hydroxyl groups. Deacylation of one orboth of the ester groups restores the ability of these compounds tofluoresce.

In one embodiment, the compounds of this invention are suitable for usein detecting cancer cells that preferentially absorb these compounds ingreater preponderance than normal cells. For example, it is literaturerecognized that ovarian, certain brain tumors, mesothelioma, breast,colon, renal, kidney and lung tumors all overexpress folic acidreceptors. Likewise, the compounds of formula II having a pteroic acidcomponent can be used in place of folic acid to assess the presence ofsuch cancer cells in a cellular composition. This is because thesecompounds are non-fluorescent and provide no background fluorescence andfurther because pteroic acid will be absorbed via the folate receptorson cancer cells. Moreover, when intracellularly absorbed into the cancercell by the folic acid receptors, intracellular enzymes such asesterases and lipases deacylate the ester group(s) of the compoundsthereby converting them from non-fluorescent to fluorescent. Such allowsthe clinician to immediately recognize the presence of such cancer cellsdue to their unambiguous fluorescence. Still further, the clinician canemploy the compounds of formula III as the glucosamine component isrecognized by glucose receptors on cancer cells.

In one embodiment, the compositions of this invention are advantageouslyused to identify remnant cancer cells after tumor resection. In thisembodiment, an aqueous composition is applied to the tissue surfaceafter resection using compounds that are preferentially absorbed bycancer cells. The cellular uptake of these compounds will result in thecancer cells becoming unambiguously fluorescent.

In one embodiment, there is also provided a method for detecting cancercells overexpressing folic acid receptors in a cellular compositionsuspected of containing such cancer cells wherein said method comprises:

-   -   selecting a compound of formula I or formula II;    -   contacting a cellular composition suspected of containing cancer        cells that overexpress folic acid receptors with said selected        compound;    -   maintaining said contact for a sufficient period of time to        permit absorption of said diester and intracellular deacylation        thereof by said cancer cells if present;    -   assessing the presence of intracellular fluorescence as an        indicia of the presence of said cancer cells.

In one embodiment, there is also provided a method for detecting cancercells in a cellular composition suspected of containing such cancercells wherein said method comprises:

-   -   selecting a compound of formula III;    -   contacting a cellular composition suspected of containing cancer        cells that preferentially absorb said compound relative to        normal cell;    -   maintaining said contact for a sufficient period of time to        permit absorption of said diester and intracellular deacylation        thereof by said cancer cells if present;    -   assessing the presence of intracellular fluorescence as an        indicia of the presence of said cancer cells.

EXAMPLES

The following non-limiting examples are provided to illustrate theclaimed invention and not to provide any limitations thereto.

In the following examples, the following terms have the followingdefinitions. If a term is undefined, it has its accepted scientificmeaning.

-   -   DCC=dicyclohexylcarbodiimide    -   DMAP=dimethylaminopyridine    -   DMF=N,N-dimethylformamide    -   eq.=equivalent    -   FBS=fetal bovine serum    -   g=grams    -   mg=milligrams    -   mL=milliliters    -   mm=millimeters    -   RPMI=Roswell Park Memorial Institute Medium    -   TLC=thin layer chromatography    -   v/v=volume to volume

Example 1—Synthesis of Methoxyethoxycarbonylproprionic Acid (Compound12) (Scheme 1)

The above reaction follows the literature preparation described by J.Materials Chemistry, 2014, 2(26):4142-4145. Specifically, a slightexcess of succinic anhydride was combined with 2-methoxyethanol inmethylene chloride in a flask at about 20° C. A solution oftriethylamine in methylene chloride was added dropwise over about a 15minute period during which the reaction produced sufficient heat so thatthe solvent began to boil. Afterwards, the addition of triethylamine wasstopped and the reaction stirred overnight after returning to roomtemperature.

The reaction was stopped and the reaction solution washed with brine andthe organic layer was recovered. The solvent was stripped and theresulting product was purified by column chromatography (silica gelusing a gradient of from 0 to 10% methanol in methylene chloride). Theresulting product (compound 10) was used as is without furtherpurification or isolation.

Example 2—Synthesis of the Fluorescein Diester, Compound 14 (Scheme 2)

Approximately 1 equivalent of compound 12 was dissolved in methylenechloride and then combined with approximately 1 equivalent of DCC atroom temperature. The mixture was stirred for approximately 5 minutesand then 0.25 equivalents of DMAP and approximately 0.25 equivalents offluorescein 5-isothiocyanate (W is —N═C═S) were added thereto. Thereaction mixture was then sonicated at 26° C. until the suspension wassubstantially dissipated which occurred over an approximate 15 minuteperiod. The resulting reaction mixture was stirred overnight at roomtemperature and monitored for reaction completion by TLC. Uponsubstantial reaction completion, the non-soluble components werefiltered and the resulting solution was placed on a silica column forpurification purposes. The column was eluted with a solvent gradientstarting at 0% methanol and 100% methylene chloride and finishing with10% methanol and 90% methylene chloride (v/v). The elutant containingthe desired compound was stripped of solvent and the resulting compound14 was substantially free of fluorescence indicative of formation ofdiester. The water solubility of the compound was evaluated and assessedto be at least 10 mg/mL. A small aliquot of the compound was contactedwith a sodium hydroxide solution that immediately provided forfluorescence indicative of deacylation.

Example 3—Synthesis of the Folic Acid—Fluorescein Diester Conjugate,Compound 23

Compound 20 was prepared according to the literature as set forth inGuaranga, et al., Bioconjugate Chemistry, 2012, 23:84-96. In particular,compound 20 was prepared from folic acid by addition of about 6equivalents of DCC in a solution of about 4:1 DMF:pyridine. The reactionwas maintained at room temperature overnight. The solution was filteredor centrifuged to removed solids and the resulting solution containingcompound 20 was combined with approximately 1 equivalent of compound 21.The reaction was maintained at room temperature overnight and theresulting solution was filtered again to remove additional solids toprovide containing compound 22.

Alternatively, the reaction can be conducted by combining folic acid,DCC and compound 21 into a single reaction to provide for compound 22where, again, solids are removed by centrifugation or filtration.

Compound 22 was optionally combined with methanol to provide for themethyl ester—compound 22 A. The Boc protecting group was removed byconventional methods and the resulting compound 22B (not shown) wascombined with compound 14 in DMF at room temperature overnight so as toprovide for compound 24.

Specifically, compound 14 was combined with compound 22B underconditions wherein the aromatic amino group reacted with theisothiocyanate to provide for compound 24.

Exemplary compounds that can be made by the methods described hereininclude the following:

Comparative Compound A each R is hydrogen

Compound 24—Each R is CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—

Compound 25—Each R is CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—

Compound 26—Each R is CH₃OCH₂CH₂OC(O)CH₂CH₂C(O)—

Biology

Compound 24 and comparative compound A were evaluated for their abilityto be absorbed by cancer cells and then, in the case of compound 24,deacylated by intracellular esterases so as to regenerate a fluorescentstructure. Specifically, approximately 500,000 SDOV3 cells (an ovariancancer cell line) were seeded into separate 35 mm culture dishescontaining a folate-free growth medium (RPMI+10% FBS—fetal bovineserum). The next day, the medium was replaced with a folate-free medium(no FBS). In one culture dish, the medium was supplemented with 25micromolar of compound 24; and, in another culture dish, the medium wassupplemented with 50 micromolar of comparative compound A. Afterincubation, the cells were washed with HBSS (Hank's balanced saltsolution) to remove unbound compound. The cells were then imaged with a20× immersion objective on a standard upright fluorescent microscope. Inthe case of compound 24, the fluorescent signal was clear, consistentand unambiguous evidencing that cancer cells were fluorescent and thatthe fluorescent signal was not evident in the solution. FIG. 1illustrates a picture showing the fluorescence generated. Note that onlythe cancer cells evidenced fluorescence and that the solution remainednon-fluorescent.

These results establish that compound 24 targeted cancer cells, wereabsorbed by cancer cells, and were deacylated by intracellular enzymes.The persistent signaling solely in the cancer cells evidenced thatdeacylated compound 24 did not efflux from the cancer cells. On theother hand, comparative compound A also was absorbed by the cancer cellsand immediately fluoresced but that was followed by loss of fluorescencelikely due to bleaching under the intense light used.

Taken together, the compounds of this invention are suitable for use indetecting remnant cancer cells. Because certain cancer cellspreferentially uptake the conjugates of this invention, after incubationfor a period of time, removal of the applied solution from the surgicalfield will limit absorption into normal cells. Such can be accomplishedunder conventional lavage/washing conditions.

What is claimed is:
 1. A method for detecting cancer cells in a cellularmass suspected of containing cancer cells which method comprisescontacting said cellular mass with a pro-fluorescent compound of formulaII:

wherein said compound emits a fluorescent fingerprint in the presence ofviable cancer cells; measuring the fluorescence obtained by exposing thecellular mass to UV light; and correlating said emitted fluorescence tosaid fluorescent fingerprint so as to assess the presence or absence ofcancer cells, further wherein R and R¹ are independently C₂-C₁₈ alkyl orcycloalkyl groups either or both optionally containing 1 to 8heteroatoms selected from the group consisting of oxygen, S(O)_(x),>NR₃, —OP(O)_(y)H, —(O)S(O)_(z)H, —C(O)—, amino, C₁-C₆-alkylamino,(C₁-C₆)-dialkylamino, —C(O)O—, —C(O)OH, and oxo wherein y and z areindependently 1 or 2, and x is 0.1 or 2; R³ is hydrogen; and L is alinker of from 1 to 20 carbon atoms and 1 to 6 heteroatoms selected fromthe group consisting of oxygen, S(O)_(x), >NR₃, —OP(O)_(y)H,—OS(O)_(z)H, —C(O)—, amino, C₁-C₆ alkylamino, (C₁-C₆)-dialkylamino,—C(O)O—, —C(O)OH, and oxo wherein y and z are independently 1 or 2, andx is 0.1 or 2; or pharmaceutically acceptable salts and/or solvatesthereof.
 2. The method of claim 1 wherein said compound, salt or solvatethereof is selected from the group consisting of: II

No. R/R¹ L  9 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—

10 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—

11 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—

12 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—/ CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—

13 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O— CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—

14 CH₃OCH₂CH₂OC(O) CH₂CH₂C(O)O—/ CH₃CH₂C(O)O—

15 CH₃O₂C(CH₂CH₂O)₃ C(O)(CH₂CH₂O)₃C (O)O—

16 CH₃O(CH₂CH₂O)₄ CH₂CH₂C(O)O—/ CH₃O(CH₂CH₂O)₄ CH₂CH₂C(O)O—